MRPS25 Antibody

What is MRPS25 and why is it significant in mitochondrial research?

MRPS25 (also known as MRP-S25, S25mt, or RPMS25) is a structural component of the 28S small subunit of the mitochondrial ribosome. Unlike many mitoribosomal proteins, MRPS25 does not have a bacterial homolog, indicating it emerged during the evolutionary divergence of mitochondrial ribosomes from their bacterial origins .

MRPS25 is essential for the assembly and stability of the entire 28S subunit. Research has demonstrated that mutations in MRPS25 can destabilize the small ribosomal subunit, leading to impaired mitochondrial translation and consequently decreased levels of oxidative phosphorylation (OXPHOS) proteins . The protein has gained clinical significance following the discovery that mutations in MRPS25 can cause mitochondrial disease characterized by dyskinetic cerebral palsy and partial agenesis of the corpus callosum .

What are the optimal applications for MRPS25 antibodies in experimental research?

MRPS25 antibodies are versatile tools that can be employed in multiple experimental applications:

When selecting an application, consider that Western blotting has been most extensively validated for MRPS25 detection, with positive results confirmed in HEK-293 and HepG2 cells . For immunohistochemistry, human intrahepatic cholangiocarcinoma and stomach tissues have shown successful staining .

How should researchers validate the specificity of MRPS25 antibodies?

Validating antibody specificity is critical for reliable experimental results. For MRPS25 antibodies, implement the following validation strategies:

• Positive and negative controls: Use cells or tissues known to express MRPS25 (such as HEK-293 or HepG2 cells) as positive controls . For negative controls, consider using MRPS25-knockdown or knockout cells.

• Molecular weight verification: Confirm that your antibody detects a band at the expected molecular weight of approximately 20 kDa in Western blot applications .

• Cross-validation with multiple antibodies: Use antibodies targeting different epitopes of MRPS25, such as N-terminal versus C-terminal regions .

• Validation in patient samples: The most stringent validation can be performed using samples from patients with confirmed MRPS25 mutations, which should show significantly reduced protein levels (approximately one-tenth of control levels for the p.P72L mutation) .

• Functional complementation: In cells with MRPS25 mutations, expression of wild-type MRPS25 should restore protein levels and function, providing further validation of antibody specificity .

What are the key considerations for Western blotting with MRPS25 antibodies?

When performing Western blot analysis with MRPS25 antibodies, consider these methodological details:

• Sample preparation: For optimal results, extract proteins from cells or tissues using buffers containing protease inhibitors to prevent degradation of MRPS25.

• Loading controls: Include appropriate mitochondrial loading controls such as VDAC or other mitochondrial proteins that are not affected by MRPS25 status.

• Detection of related proteins: Consider simultaneously probing for other components of the 28S subunit (such as MRPS17, MRPS22, and MRPS29) and the large mitochondrial ribosomal subunit (such as MRPL44 and MRPL45) to assess the broader impact of experimental conditions on mitoribosomal integrity .

• Quantification: In Western blot analysis of patient fibroblasts with MRPS25 mutations, steady-state levels of MRPS25 were observed to be approximately one-tenth of control levels, providing a benchmark for expected changes in pathological conditions .

• Secondary antibodies: For rabbit polyclonal MRPS25 antibodies, appropriate secondary antibodies include goat anti-rabbit IgG conjugated with HRP, AP, biotin, or FITC .

What disease-associated mutations have been identified in MRPS25 and how can antibodies help study them?

A homozygous c.215C>T variant in MRPS25, resulting in a p.P72L substitution, has been identified in a patient with dyskinetic cerebral palsy and partial agenesis of the corpus callosum . This mutation affects a highly conserved proline residue and compromises inter-protein contacts, destabilizing the small ribosomal subunit.

Antibodies are invaluable tools for studying such mutations:

• Protein level assessment: Western blotting with MRPS25 antibodies revealed that the p.P72L mutation reduces MRPS25 protein levels to approximately one-tenth of normal levels .

• Effect on associated proteins: Using antibodies against multiple mitoribosomal proteins demonstrated that the p.P72L mutation also reduces levels of other 28S subunit components (MRPS17, MRPS22, and MRPS29) while 39S subunit components (MRPL44 and MRPL45) remained unaffected .

• Complementation studies: Antibodies can verify the restoration of MRPS25 and associated protein levels following lentiviral-mediated expression of wild-type MRPS25 in mutant cells, confirming the pathogenicity of identified variants .

• Diagnostic applications: Antibodies against MRPS25 could potentially serve as diagnostic tools in muscle biopsies from patients with suspected mitochondrial translation defects.

How can researchers analyze the impact of MRPS25 deficiency on mitochondrial ribosome assembly?

Mitochondrial ribosome assembly can be assessed using several approaches:

• Sucrose gradient analysis: Patient fibroblasts with MRPS25 mutations show scarce assembled 28S subunits when analyzed on sucrose gradients, with MRPS components concentrated at the top of the gradient among soluble and individual polypeptides . This technique can reveal:

  • Distribution of 28S and 39S subunits

  • Presence of fully assembled 55S ribosomes

  • Shifts in buoyant density of ribosomal components

• Analysis of ribosomal RNA levels: Quantitative analysis showed that in cells with MRPS25 mutations, 12S rRNA (component of the 28S subunit) was reduced to approximately 60% of control values, while 16S rRNA (component of the 39S subunit) was increased to 1.5 times the control value .

• Immunoprecipitation of ribosomal complexes: Anti-MRPS25 antibodies can be used to pull down associated ribosomal components, allowing for the characterization of protein-protein interactions within the ribosomal complex.

• Complementation verification: Following introduction of wild-type MRPS25 into cells with MRPS25 mutations, antibodies can confirm restoration of normal ribosomal assembly profiles on sucrose gradients .

What methodological approaches can be used to study mitochondrial translation in the context of MRPS25 dysfunction?

Several experimental approaches can assess mitochondrial translation in MRPS25-deficient systems:

• Metabolic labeling of mitochondrial translation products: Pulse-chase experiments with radiolabeled amino acids in the presence of cytosolic translation inhibitors can directly measure mitochondrial protein synthesis rates and patterns.

• Analysis of OXPHOS protein levels: Western blotting with antibodies against mitochondrially-encoded respiratory chain components can serve as a proxy for mitochondrial translation efficiency. In cells with MRPS25 mutations, decreased levels of multiple respiratory chain subunits have been observed .

• Complementation studies: Transgenic expression of wild-type MRPS25 in mutant cells resulted in partial restoration of OXPHOS protein levels, confirming the causal relationship between MRPS25 dysfunction and impaired mitochondrial translation .

• Ribosome profiling: This technique can provide insight into the positions of ribosomes on mRNAs, potentially revealing translation stalling or inefficiency caused by MRPS25 deficiency.

How do MRPS25 antibodies perform across different species for comparative studies?

Different commercially available MRPS25 antibodies show varying cross-reactivity patterns:

When conducting comparative studies across species, consider:

• Epitope conservation: Antibodies targeting highly conserved regions (such as C-terminal epitopes) generally show better cross-reactivity across species.

• Validation in each species: Even when manufacturers claim cross-reactivity, independent validation in each species of interest is recommended.

• Control samples: Include positive controls from each species being studied to confirm reactivity and determine optimal working dilutions.

What are the technical considerations for immunohistochemical detection of MRPS25?

For successful immunohistochemical detection of MRPS25:

• Antigen retrieval: For FFPE tissues, heat-induced epitope retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may serve as an alternative .

• Antibody dilution: Start with a 1:50-1:500 dilution range and optimize based on signal-to-noise ratio .

• Tissue selection: MRPS25 has been successfully detected in human intrahepatic cholangiocarcinoma and stomach tissues .

• Controls: Include tissues known to express high levels of MRPS25 as positive controls, and consider using tissue from patients with confirmed MRPS25 mutations as negative or reduced expression controls when available.

• Counterstaining: Given MRPS25's mitochondrial localization, counterstaining with mitochondrial markers can help confirm proper subcellular localization and specificity.

How can researchers design knockout or knockdown experiments to study MRPS25 function?

When designing genetic manipulation experiments to study MRPS25:

• CRISPR/Cas9 knockout considerations: Complete knockout of MRPS25 may be lethal, as suggested by the observation that even patient fibroblasts with the p.P72L mutation retain approximately 10% of normal MRPS25 levels . Consider:

  • Inducible knockout systems

  • Tissue-specific knockouts in animal models

  • Heterozygous knockout approaches

• siRNA/shRNA knockdown: Titrate knockdown levels to achieve different degrees of MRPS25 reduction. This approach can help establish dose-response relationships between MRPS25 levels and:

  • Mitoribosome assembly

  • Mitochondrial translation rates

  • OXPHOS complex formation

  • Cellular phenotypes

• Rescue experiments: Design complementation studies with wild-type or mutant MRPS25 constructs to verify phenotype specificity. Note that overexpression of MRPS25 in normal cells resulted in marked cell death, suggesting a possible toxic effect in the context of normal 28S subunit assembly .

• Readouts: Measure multiple parameters to comprehensively assess the impact of MRPS25 manipulation:

  • Mitoribosome assembly using sucrose gradients

  • Mitochondrial translation using metabolic labeling

  • OXPHOS complex levels and activity

  • Mitochondrial morphology and function

  • Cellular growth and viability

What proteomics approaches can reveal the broader impact of MRPS25 dysfunction?

Advanced proteomics methods can provide comprehensive insights into how MRPS25 dysfunction affects the mitochondrial proteome:

• Quantitative proteomics: In patients with MRPS25 mutations, quantitative proteomics revealed:

  • ~4.5-fold reduction in MRPS25 levels

  • Coordinated reduction in other MRPSs

  • Differential effects on large versus small ribosomal subunit components

• Relative Complex Abundance analysis: This sensitive method can identify defects in OXPHOS disorders with similar or greater sensitivity than traditional enzymology, and can be applied to detect alterations in ribosomal complex assembly caused by MRPS25 deficiency .

• Interaction proteomics: Immunoprecipitation of MRPS25 followed by mass spectrometry can identify protein-protein interactions and how they are affected by disease-causing mutations.

• Post-translational modification analysis: Examine how MRPS25 dysfunction affects the post-translational modification landscape of mitochondrial proteins, potentially revealing compensatory mechanisms or pathological changes.

How can researchers differentiate between primary MRPS25 defects and secondary mitoribosomal abnormalities?

Distinguishing primary MRPS25 defects from secondary mitoribosomal abnormalities requires a systematic approach:

• Genetic analysis: Identify potentially pathogenic variants in MRPS25 through exome or genome sequencing.

• Protein level assessment: Use Western blotting with MRPS25 antibodies to determine if MRPS25 levels are specifically reduced compared to other mitoribosomal proteins.

• Ribosomal assembly profiling: In MRPS25 mutations, a characteristic pattern emerges:

  • Severely reduced assembled 28S subunits

  • Relatively normal 39S subunits, albeit with subtle changes in buoyant density

  • Accumulation of unassembled MRPS components at the top of sucrose gradients

• rRNA analysis: A specific pattern of reduced 12S rRNA with elevated 16S rRNA is associated with MRPS25 mutations .

• Complementation studies: Restoration of phenotypes by wild-type MRPS25 expression confirms primary MRPS25 deficiency .

• Immunofluorescence colocalization: Examine colocalization of MRPS25 with other mitoribosomal markers to assess integration into ribosomal structures.

What are the future directions for MRPS25 antibody development and applications?

Several promising avenues exist for advancing MRPS25 antibody technology:

• Development of monoclonal antibodies: Most current MRPS25 antibodies are polyclonal; monoclonal antibodies could provide greater specificity and reproducibility for research and potential diagnostic applications.

• Antibodies against specific MRPS25 mutations: Developing antibodies that specifically recognize mutant forms of MRPS25 could aid in fundamental research and potentially diagnostics.

• Proximity labeling applications: MRPS25 antibodies conjugated with enzymes for proximity labeling could help map the protein's immediate neighborhood within the mitoribosome.

• Super-resolution microscopy: Next-generation MRPS25 antibodies optimized for super-resolution microscopy could provide unprecedented insights into mitoribosome organization within mitochondria.

• Therapeutic applications: While currently theoretical, antibody-based approaches to detect or manipulate MRPS25 levels could eventually have therapeutic applications for mitochondrial disorders.

How do mutations in MRPS25 compare with mutations in other mitoribosomal proteins?

Understanding the similarities and differences between MRPS25 mutations and other mitoribosomal protein mutations provides important context:

• Clinical presentations: Mutations in different mitoribosomal proteins often present with overlapping but distinct clinical features. MRPS25 mutations have been associated with dyskinetic cerebral palsy and partial agenesis of the corpus callosum , while other MRP mutations may present differently.

• Molecular consequences: MRPS25 mutations primarily affect the small ribosomal subunit, with indirect effects on the large subunit . In contrast, mutations in proteins like MRPL39 directly destabilize the large mitoribosomal subunit .

• Complementation patterns: When introducing wild-type MRPS25 into control cells, marked cell death was observed, suggesting a possible toxic effect in the context of normal 28S subunit assembly . This pattern may differ from other mitoribosomal proteins.

• Evolutionary conservation: MRPS25 is one of 15 structural subunits of the mitochondrial 28S that does not have a bacterial homolog , which may influence its functional redundancy compared to more evolutionarily conserved components.

What methodological approaches can detect subtle conformational changes in MRPS25?

Detecting conformational changes in MRPS25, particularly those induced by mutations like p.P72L, requires sophisticated techniques:

• Structural studies: The high-resolution structure of the 28S ribosome provided insights into how the p.P72L mutation compromises inter-protein contacts and destabilizes the small subunit . Similar approaches can be applied to study other mutations.

• Conformation-specific antibodies: Developing antibodies that recognize specific conformational states of MRPS25 could help track structural changes under different conditions.

• Hydrogen-deuterium exchange mass spectrometry: This technique can reveal dynamic structural changes in MRPS25 in its free form versus when incorporated into the 28S subunit.

• Crosslinking studies: Chemical crosslinking followed by mass spectrometry can map distance constraints between MRPS25 and neighboring proteins, revealing how mutations alter these interactions.

• Molecular dynamics simulations: In silico approaches can predict how specific mutations affect MRPS25 conformation and stability, guiding experimental studies.